Acta Herpetologica 11(1): 85-89, 2016 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/Acta_Herpetol-16491 Introduction of Eleutherodactylus planirostris (Amphibia, Anura, Eleutherodactylidae) to Hong Kong Wing Ho Lee1, Michael Wai-Neng Lau2, Anthony Lau3, Ding-qi Rao4, Yik-Hei Sung5,* 1 Department of Life Science, Tunghai University, Taichung 407, Taiwan 2 WWF-Hong Kong, Kwai Chung, Hong Kong SAR, China 3 School of Biological Sciences, University of Hong Kong, Pokfulam Road, Hong Kong SAR, China 4 Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming 650223, Yunnan, China 5 Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China. *Corresponding author. E-mail: yhsung@hkbu.edu.hk Submitted on 2015, 13th August; revised on 2016, 7th January; accepted on 2016, 9th January Editor: Uwe Fritz Abstract. An unidentified small frog species was first encountered in Hong Kong Special Administration Region (SAR), China, in 2000, where the local amphibian diversity is well-studied. We herein identified this unknown frog as Eleutherodactylus planirostris (greenhouse frog) using DNA barcoding. We found that its distribution in Hong Kong is widespread (>18 localities), and breeding has been observed in multiple occasions. The populations in at least four localities persisted for over seven years. We discuss its potential negative impacts to terrestrial ecosystems in Hong Kong, with particular concern of its potential competition with the endemic Liuixalus romeri. We call for studies to investigate the impacts of the introduced E. planirostris on the local ecosystem. Screening for E. planirostris in export- ed plants from Hong Kong should be carried out. Keywords. Amphibians, biological invasions, greenhouse frog, Liuixalus romeri. The amphibian diversity of Hong Kong, is well stud- ied (Lau, 1999). Owing to the subtropical climate, altitu- dinal range (highest peak at 957m) and variable terrain it contains a rich mixture of habitats. As a result the high- ly urbanized Hong Kong is home to a surprisingly high species richness of amphibians [23 species of frogs and 1 species of salamander (Chan et al., 2005)]. In 2000, a morphologically distinct frog not assigned to any of the known native species was captured by the first author in a container yard at Hung Shui Kiu in Hong Kong (Fig 1, Table 1). Subsequently, additional frogs with similar mor- phology have been found at 18 localities between 2002 and 2015. In Asia-Pacific region, E. planirostris has been recorded in Guam (Christy et al., 2007a) and the Phil- ippines (Olson et al., 2014; Sy et al., 2015; Sy and Saigo, 2015); to our knowledge, this is the first reported case of E. planirostris introduction and establishment in conti- nental Asia. This study was conducted in Hong Kong Special Administrative Region, China. Distribution data of this frog species was compiled from region-wide opportunistic surveys between 2000 and 2015. Geographic locations of where this species occurred were recorded using a hand- held GPS unit. Two adult specimens were collected from Wu Kau Tang in 2013 for molecular analysis and were deposited at The Museum of Biology, Sun Yat-sen Uni- versity (Museum voucher number: SYS a004514 and SYS a004515). Genomic DNA from the two specimens was extracted using a DNA extraction kit (FavorPrep Tissue Genomic DNA Extraction Mini Kit, Favorgen Biotech Corporation, Ping-Tung, Taiwan) following manufacturer’s protocol and a 562-bp fragment of ribosomal 16S gene was 86 Wing Ho Lee et alii amplified using Polymerase Chain Reaction. The resulting products were sequenced on an ABI Prism 3730 automat- ed DNA sequencer and the results were submitted to the program BLAST to identify similar species from the Gen- Bank database and program MOLE-BLAST to produce phylogenetic tree. Genetic sequences have been deposited in GenBank (GenBank accession number KM252679 and KM252680). A study has since been carried out to con- firm the identity of this unknown species and to docu- ment its distribution in Hong Kong. The unknown frog has been identified as Eleutherodactylus planirostris (green- house frog), a direct-developing Eleutherodactylid native to a number of Caribbean islands (Bahamas, Cayman Islands and Cuba) (Dodd, 2013). It has been accidentally introduced to many countries throughout the New World (Olson et al., 2012; Andrew et al., 2011; Heinicke et al., 2011), and Asia-Pacific Islands including Hawaii (Kraus, 1999), Guam (Christy et al., 2007a) and the Philippines (Olson et al,. 2014; Sy et al., 2015; Sy and Saigo, 2015), most likely via the live plant trade. The sequences of the frogs collected from Wu Kau Tang had 100% max identity with the sequences of E. planirostris collected at Naples, Collier, Florida (GenBank accession number: DQ283107; Fig. 2) (Frost et al., 2004). Four sites (Aberdeen, Pokfulam Country Park, Tai Lam Country Park and Tai Tong) were revisited between 2014 and 2015 and robust E. planirostris populations were observed at all four sites. E. planirostris appear to be habitat generalist in Hong Kong, occupying a diversity of habitats including second- ary forests, shrubland, agricultural fields, near fishponds, urban parks and near human settlements such as village houses and container yards. We found 22 eggs on wet leaf litter in Aberdeen in May 2006 and the eggs hatched after two weeks in captivity. Egg masses were also found in Pokfulam Country Park and Tai Tong in July 2007. Fig. 1. Locations where Eleutherodactylus planirostris were found in Hong Kong. Letters identify the localities, as presented in Table 1. The grey area indicates natural range of Liuixalus romeri. Asterisks indicates distribution localities of E. planirostris that overlap with transloca- tion sites of L. romeri. 87Introduction of Eleutherodactylus planirostris to Hong Kong Several individuals kept in captivity from 2009 to 2011 readily ate small crickets, spiders, termites and fruit flies. They produced egg clutches containing 11-17 eggs. E. planirostris feed on invertebrates including insects, spiders and snails (Global Invasive Species Database, 2010). In Hong Kong, E. planirostris is sympatric with eight species of anurans (Duttaphrynus melanostictus, Fejervarya limnocharis, Hoplobatrachus rugulosus, Kaloula pulchra, Liuixalus romeri, Megophrys brachykolos, Odor- rana chloronota and Polypedates megacephalus). However, little is known about the diet of E. planirostris in the wild in Hong Kong and the population status of terrestrial invertebrates in Hong Kong, thus its impacts on native invertebrate populations remain unknown. In the originally amphibian-free Hawaii, introduced E. planirostris can exist in high density, where it com- petes for food with other insectivores (Olson et al., 2012; Beard et al., 2009; Beard and Pitt, 2005) including birds (Global Invasive Species Database 2010). The congener- ic Eleutherodactylus coqui is listed as one of the world’s worst invasive alien species by IUCN (Lowe et al., 2000). On the ecosystem level, the introduction of E. coqui can alter ecosystem functioning by distorting nutrient cycling in Hawaii (Sin et al., 2008), however the impacts caused by E. coqui can be outweighed by that caused by invasive plants (Tuttle et al., 2009). The impact of E. planirostris’s introduction on the endemic L. romeri is of potential concern. As adult E. planirostris are smaller than most of the frog species in Hong Kong, we speculate that resource competition will be most intense with the sim- ilar-sized, small bodied L. romeri (Lau and Zhao, 2004). Competition between E. planirostris and L. romeri might be significant because (1) their sizes are similar and E. planirostris consume arthropods (Olson et al., 2012) that overlap broadly with the natural preys of L. romeri (Lau, 1998) which indicates a potentially high trophic similar- ity and (2) we observed E. planirostris dwell on the for- est floors in secondary forests and forest edges, where L. romeri inhabit. We found E. planirostris at Tai Lam Coun- try Park and Kadoorie Farm and Botanic Garden, where populations of L. romeri were translocated which was a mitigation measures of the construction of the Chek Lap Kok airport in 1990s (Lau and Banks, 2008). E. planiro- stris has yet to be found in the natural range of L. romeri, including Chek Lap Kok, Lamma Island, Lantau Island and Po Toi Island (Lau, 1999; Fig. 1). Monitoring of E. planirostris should be carried out in the L. romeri range and further studies are urgently needed to understand the Table 1. Distribution of Eleutherodactylus planirostris and the respective year of discovery in Hong Kong. Site label Locations GPS coordinates Year of discovery A Hung Shui Kiu 22°25.62’N, 113°59.59’E 2000 B Tai Tong 22°25.25’N, 114°1.33’E 2002 C Aberdeen Reservoir 22°15.34’N, 114°9.59’E 2006 D Pokfulam Country Park 22°16.03’N, 114°8.34’E 2007 E Hei Ling Chau 22°15.41’N, 114°1.90’E 2007 F Shek Kong 22°25.46’N, 114°6.38’E 2007 G Tai Lam Country Park 22°24.15’N. 114°2.19’E 2007 H Chai Wan 22°15.53’N, 114°13.65’E 2008 I Tai Shue Wan 22°14.19’N, 114°9.96’E 2010 J University of Hong Kong 22°16.88’N, 114°8.24’E 2010 K Shing Mun Country Park 22°22.83’N, 114°8.56’E 2011 L Kadoorie Farm and Botanic Garden 22°25.98’N, 114°7.06’E 2011 M Nam Fung Road 22°15.26’N, 114°10.68’E 2012 N Shek Kip Mei 22°19.88’N, 114°10.09’E 2012 O Wu Kau Tang 22 30.33’N, 114 14.63’E 2012 P Long Valley 22°30.64’N, 114°6.768’E 2012 Q Wong Nai Chung Reservoir 22°15.44’N, 114°11.75’E 2013 R Tai Tam Country Park 22°15.54’N, 114°12.11’E 2015 Fig. 2. Phylogenetic tree derived from partial fragments of 16S rRNA genes using fast minimum evolution method produced by program Mole-Blast. 88 Wing Ho Lee et alii impacts of the introduced E. planirostris to the L. romeri. Our results demonstrate that E. planirostris have established in Hong Kong. The populations at several sites, i.e., Aberdeen, Pokfulam Country Park, Tai Lam Country Park, Tai Tong, persisted for at least seven years. The reproduction of E. planirostris is successful in Hong Kong because (1) it is a direct-developing species (i.e. no aquatic larval stage) (Goin, 1947), and (2) the suit- able subtropical climate of Hong Kong [annual mean temperature = 22.8oC; mean annual precipitation = 2214 mm (Dudgeon and Corlett, 2004)] that matches the cli- mate niche of the species (Rödder and Lötters, 2010). Numerous control methods have been carried out to con- trol E. coqui populations in Hawaii, and are expected to have similar effectiveness on E. planirostris (Olson et al., 2012). It is noteworthy that control methods for E. coqui are only effective in eradicating small and isolated popu- lations (Beard and Pitt, 2005). If E. planirostris is found to have significant ecological impacts in Hong Kong, prompt control and eradication methods should be exe- cuted before the populations become too large. It is likely that E. planirostris was introduced acci- dentally to Hong Kong via the live plant trade, which was similar to the introduction to Hawaii and Guam (Kraus et al., 1999; Christy et al., 2007b). Large volume of live plants was imported to Hong Kong from continental United States of America in early 2000s when E. planirostris was first detected in Hong Kong (Census and Statistics Depart- ment, Hong Kong SAR, 2001). This was supported by one frog being found in a potted Tillandsia cyanea plant bought from the flower market in Mong Kok in urban Kowloon in 2011 (Louis Fung, per. comm.) and another frog found in an apartment on Ap Lei Chau in 2015 that was likely to arrive together with an indoor plant. In addi- tion, we made observations of high densities of frogs (> 30 in 200 m2) near newly renovated slopes where nursery plants were planted on. Due to the extensive trade of live plants in the region, it seems it is a matter of time that E. planirostris will spread to other places that import plants from Hong Kong. In 2015, Hong Kong exported or re- exported over 100,000 kg of plants or parts of plants to tropical or subtropical countries/cities, including Australia, China, Macau, Malaysia, Singapore, Thailand, Taiwan and Vietnam, where the climate may be favorable to the colo- nization of E. planirostris (Census and Statistics Depart- ment, Hong Kong SAR, 2015). Screening for E. planiro- stris, including adults and eggs, in exported or re-exported plants from Hong Kong should be carried out. The small size and variations in coloration of E. planirostris as well as morphological similarity with cer- tain Asian species (e.g., Liuixalus sp. and Philautus sp.) prevented identification before molecular approach was applied to confirm its identity (Armstrong and Ball, 2005). This delayed proper measures to be taken to con- trol this introduced species. Prompt molecular analysis to identify suspicious alien species should be encouraged. We also hope this paper will help arouse the attention on this introduced frog in Asia. ACKNOWLEDGMENTS We are grateful to Louis Fung, Billy Hau, Pan Lau, Angie Ng and Philip Yip for providing useful information. REFERENCES Armstrong, K., Ball, S. (2005): DNA barcodes for biose- curity: invasive species identification. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 360: 1813-1823. Beard, K.H., Pitt, W.C. 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